G-protein-coupled receptors, such as the adrenergic receptors, elevate cellular levels of second messengers like cyclic-adenosine monophosphate (cAMP) and diacylglycerol thereby regulating and coordinating cellular metabolism and function. In the heart, β-adrenergic receptors (β-ARs), responsive to the sympathetic neurotransmitter norepinephrine and the adrenal medullary hormone epinephrine, stimulate adenylyl cyclase, raising myocardial cAMP and increasing cardiac contractility. Elevated circulating catecholamines and myocardial β-AR stimulation represent critical mechanisms for augmenting cardiac function during stress.
As is true for most G protein-coupled receptors, prolonged agonist exposure of β-ARs leads to a rapid decrease in responsiveness. Agonist-dependent desensitization can be initiated by phosphorylation of activated receptors by members of the G protein-coupled receptor kinase (GRK) family (W. P. Hausdorff et al., FASEB J. 4, 2881 (1990); J. Inglese et al., J. Biol. Chem. 268, 23735 (1993)). Phosphorylated receptors then interact with arresting proteins like β-arrestin to which they bind thereby sterically interdicting further coupling to G proteins (W. P. Hausdorff et al., 1990; J. Inglese et al., 1993). The β-adrenergic receptor kinase-1 (βARK1) is a GRK which has been shown to specifically phosphorylate activated β2-ARs in vitro and which is hypothesized to phosphorylate β2-ARs in vivo leading to uncoupling and desensitization (W. P. Hausdorff et al., 1990; J. Inglese et al., 1993; M. J. Lohse et al., Proc. Natl. Acad. Sci. U.S.A. 86, 3011 (1989); M. J. Lohse et al., J. Biol. Chem. 265, 3202 (1990); S. Pippig et al., J. Biol. Chem. 268, 3201 (1993)).
The action of βARK1 on the β1-AR has not yet been documented. βARK1 is specifically targeted to activated receptors in the plasma membrane by a translocation event mediated via a specific protein-protein interaction between the carboxyl terminus of the kinase and the βγ subunits of activated and dissociated G proteins (J. Pitcher al., Science 257, 1264 (1992); W. J. Koch et al., J. Biol. Chem. 268, 8256 (1993)).
In chronic congestive heart failure, an illness affecting more than four million Americans, there is dramatic impairment of the myocardial β-AR system. Failing human ventricular myocardium contains 50% fewer β-ARs and shows parallel decreases in agonist-stimulated adenylyl cyclase activity and even greater decreases in agonist-mediated inotropy (M. R. Bristow et al., N. Engl. J. Med. 307, 205 (1982), M. R. Bristow et (1990)). In addition, increases in inhibitory G-protein and G-protein receptor kinases (e.g. β-adrenergic receptor kinase) in heart failure may further impair receptor-mediated inotropy (T. Eschenhagen et al., Circulation Research 70, 688 (1992) and M. Ungerer et al., Circulation v7, 454 (1993)). Therapeutic interventions, involving the administration of agonists to stimulate the β-AR/adenylyl cyclase systems have an inherently limited efficacy given the reduction in receptor targets in the diseased myocardium.
An additional possible contributor to the decreased myocardial β-AR responsiveness seen in chronic failing human hearts is that levels of βARK1 are elevated (M. Ungerer et al., Circulation 87, 454 (1993); M. Ungerer et al., Circ. Res. 74, 206 (1994)). Thus, β-AR impairment in heart failure may have several underlying causes.
The field of transgenic technology has achieved significant advances in techniques for in vivo gene transfer in recent years (T. Ragot et al., Nature 361, 647 (1993); M. A. Rosenfeld et al., Cell 68, 143 (1992); R. J. Guzman, et al., Circulation Research 73, 1202 (1993)).
While several transgenic mice have been reported which express, for example, the c-myc proto-oncogene or SV-40 T-antigen affecting cardiac growth (J. L. Swain et al., Cell 50, 719 (1987); L. J. Field, Science 239, 1029 (1988); E. B. Katz et al., Am. J. Physiol. 262, H1867 (1992)), to date there have been no reports concerning the ability of a transgene to affect myocardial contractility.